Cell viability assay

Cell proliferation was determined by CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega, Fitchburg, WI, USA). Cells (3,000/well) were seeded in 96-well plates and then the medium in the wells was replaced with the medium containing diluted drug solutions of trastuzumab, lapatinib, dasatinib or their combinations in a 4-log range or complete medium, which were distributed in 8-replicate wells. Cells were incubated in the presence of each concentration of the respective drugs for 72 hours at 37°C in a humidified atmosphere of 5% CO₂ in air. After that, MTS dye was added to each well. The cultures were incubated for another 1 hour at 37°C in a humidified atmosphere with 5% CO₂. Optical densities of samples were measured at 492 nm using Multiskan^™ FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA). Mean optical density at each drug concentration was calculated after discarding the highest and lowest values. The anti-tumor effects of respective drugs for each cell line were shown in terms of inhibitory concentration at 25% (IC₂₅) for trastuzumab or 50% (IC₅₀) lapatinib, which was determined by plotting the graph of percentage of cell growth inhibition (Y-axis) versus drug concentration (X-axis). IC₂₅ and IC₅₀ values were expressed as mean and standard errors (SE). The assays were repeated more than three times.